Influenza A virus is a respiratory pathogen that causes annual epidemics and sporadic pandemics (Wright et al., 2013). Moreover, highly pathogenic avian H5N1 and the recently emerged H7N9 influenza viruses have caused an appreciable number of human infections with high mortality rates (Watanabe et al., 2013; Zhang et al., 2013). Influenza viruses infect respiratory epithelial cells and alveolar macrophages in mammalian hosts (Yu et al., 2010). The host immune system recognizes the RNA genome of influenza viruses via cytosolic sensors (Diebold et al., 2004; Pichlmair et al., 2006), which trigger innate immune responses that lead to the production of type I interferons (IFNs) and proinflammatory cytokines and chemokines (Honda and Taniguchi, 2006). Type I IFNs upregulate the production of antiviral proteins including myxovirus resistance (Mx), oligoadenylate synthetase (OAS), and interferon-stimulated gene 15 (ISG15) (Garcia-Sastre et al., 2011). Dysregulation of the innate immune responses to influenza virus infection causes lung pathology mediated by infiltrating immune cells, including macrophages and neutrophils (Heron et al., 2008; Perrone et al., 2008). Although several studies have addressed host responses to influenza virus infections (Fakuyama and Kawaoka, 2011), the mechanisms of influenza virus-induced pathology are still not fully understood.
To analyze the immune responses to influenza virus infection in vivo, viruses have been generated that expressed a fluorescent reporter protein (Kittel et al., 2004; Shinya et al., 2004). However, these viruses were significantly attenuated (Kittel et al., 2004; Shinya et al., 2004) and may not accurately reflect natural infections. For example, Manicassamy et al. (2010) generated a GFP-expressing influenza virus, which they used to assess the route of antigen presentation upon influenza virus infection (Helft et al., 2012). However, the GFP gene was not stably maintained during replication in mouse lung or cultured cells (Manicassamy et al., 2010).
Highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype continue to evolve in nature, threatening animal and public health. These viruses were first identified in Guangdong province in China in 1996 (Li et al., 2006), and have since been found in over 63 countries in multiple avian species, repeatedly infecting mammals such as pigs and humans (Li et al., 2010; Neumann et al., 2010). By December 2013, 648 human cases of H5N1 virus infection had been confirmed by the World health organization (WHO), of which 384 were fatal, yielding a mortality rate of almost 60% (http://www.who.int). In addition, novel subtypes of influenza viruses, such as H7N9 and H10N8 virus, have spontaneously appeared and sporadically infected humans causing fatal outcomes (Chen et al., 2014; Li et al., 2013) (http://www.who.int). Thus, the current threat from influenza viruses reminds us of the urgent need to gain a thorough understanding of their pathogenic mechanism in order to develop more effective strategies for control, including dynamic processes of influenza virus infection and virus-target cells in vivo remain unclear.